Transaminase-PLP co-immobilized enzyme and preparation and application thereof

A technology of co-immobilization of enzymes and transaminases, applied in the fields of application, transferase, biochemical equipment and methods, etc., can solve the problems of less recovery batches, poor stability, high temperature, etc., and achieve high product yield and purity, organic solvent Strong tolerance, the effect of reducing the discharge of "three wastes"

Active Publication Date: 2019-05-21
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2010, Dominik prepared immobilized transaminase by sol-gel / Celite 545 immobilized transaminase (embedding method). After repeated use of 8 batches, the enzyme activity remained 78%, the amount of immobilized enzyme was less, and the number of recovered batches was less. Unable to achieve industrial application (Dominik et al., 2010, Journal of Molecular Catalysis B: Enzymatic)
But when it prepares immobilized carrier, used temperature is higher and reagent (concentrated hydrochloric acid) is more dangerous and preparation step is more loaded down with trivial details; Reaction is carried out to after 13 batches, enzyme activity drops to 50%; et al., 2016, Biotechnology Reports)
[0005] At present, there are few studies on the immobilization of transaminases and the preparation of chiral amine intermediates including sitagliptin at home and abroad. The related researches have low recovery rate of enzyme activity, high preparation cost of immobilized enzyme, unsatisfactory catalytic activity, stable Poor performance and other problems seriously limit the application of immobilized biocatalysts in the production of chiral amine drug intermediates
And the report that is used for the biosynthesis of sitagliptin all needs to add extra coenzyme PLP, has so just further increased production cost, has increased the difficulty of suitability for industrialized production

Method used

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  • Transaminase-PLP co-immobilized enzyme and preparation and application thereof
  • Transaminase-PLP co-immobilized enzyme and preparation and application thereof
  • Transaminase-PLP co-immobilized enzyme and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Pretreatment of resin

[0038] a. Epoxy resin pretreatment: take 100g of epoxy resin LX-1000HFA (purchased from Xi'an Lanxiao Science and Technology New Materials Co., Ltd.), soak it in 500mL distilled water for 12h, and then suction filter to obtain the filter cake, which is the pretreated resin, Store at 4°C until use.

[0039] b, amino resin pretreatment:

[0040] ①Solution preparation:

[0041] 0.1M pH 8.0 buffer solution (1L): Add 23.8g of dipotassium hydrogen phosphate and 2.75g of potassium dihydrogen phosphate to 1L of deionized water, dilute to 1000mL, and adjust the pH to 7.8-8.2.

[0042] 2% glutaraldehyde phosphate buffer solution (1L): 40 mL glutaraldehyde (50%), 960 mL water, 4.76 g of dipotassium hydrogen phosphate, after dissolving, adjust the pH to 7.8-8.2 with potassium dihydrogen phosphate.

[0043] ②Vector activation:

[0044] Add 100 mL of 0.1M pH 8.0 buffer to 10 g of the carrier. After constant temperature shaking at 25°C and 300 rp...

Embodiment 2

[0047] Example 2: Cultivation of transaminase genetically engineered bacterial cells

[0048] (1) Construction of transaminase genetically engineered bacteria:

[0049] Primer 1 (CCG 1) was designed according to the gene sequence SEQ ID NO. CATATG GCTATCA TCCAGGTTCAGC), primer 2 (TTG CTCGAG TCAAGCCGGA ACAGAAGAG), and Nde I and Xho I restriction sites (underlined) were introduced in primer 1 and primer 2, respectively. Under the initiation of primer 1 and primer 2, high-fidelity PfuDNA polymerase was used for amplification, and the recombinant plasmid pMD18-T-BgTA ( figure 1 ) as a template, obtain the transaminase BgTA gene sequence, utilize Nde I and Xho I restriction endonucleases (TaKaRa) to process the amplified fragment after sequencing, and utilize T4 DNA ligase (TaKaRa) to use the same restriction for this fragment The endonuclease-treated commercial vector pET28b (Invitrogen) was ligated to construct the expression vector pET28b-BgTA ( figure 2 ). The construct...

Embodiment 3

[0052] Example 3: Preparation of crude transaminase enzyme solution

[0053] The cells were suspended in pH 9.0 Tris buffer according to the concentration (100 g / L), and the cells were disrupted with a high-pressure homogenizer under a pressure of 30 Mpm. After crushing, the crude enzyme liquid is centrifuged at 9000 rpm for 10 min, and the supernatant is taken after centrifugation, which is the crude transaminase enzyme liquid.

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Abstract

The invention relates to a transaminase-PLP co-immobilized enzyme and a preparation and application thereof, and application of the co-immobilized transaminase for preparing a Sitagliptin medicine chiral midbody. Epoxy resin is adopted as a carrier, and the transaminase-PLP co-immobilized enzyme is obtained by covalence immobilizing of transaminase and coenzyme pyridoxal phosphate (PLP for short).The transaminase-PLP co-immobilized enzyme is adopted as a catalyst, the stability is good, the service life is long, the organic solvent tolerance is good, the transaminase-PLP co-immobilized enzymecan be repeatedly used, no expensive exogenous coenzymes are added in the reaction process, and the production cost is greatly lowered. The method is simple in technology, low in cost, and high in product yield and purity, and the enzyme has the great application value in industrial production of the Sitagliptin medicine chiral midbody.

Description

(1) Technical field [0001] The invention relates to a transaminase-PLP co-immobilized enzyme and a preparation method thereof, as well as its application in preparing a sitagliptin intermediate by asymmetric reduction. (2) Background technology [0002] Diabetes is one of the three major non-communicable chronic diseases alongside cardiovascular and malignant tumors, seriously endangering human health and social development. The World Health Organization first released the Global Diabetes Report in 2016, showing that the number of adults with diabetes worldwide has tripled in the past 40 years, most of whom live in developing countries. In 2014, there were 422 million people with diabetes in the world over the age of 18, accounting for 8.5% of the world's total population. The prevalence of diabetes among Chinese adults is close to 10%. Sitagliptin is a new type of dipeptidyl peptidase-IV (DPP-4) inhibitor drug, which can effectively treat type II diabetes. Compared with t...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/70C12P17/18
Inventor 柳志强张晓健郑裕国范浩浩程峰贾东旭
Owner ZHEJIANG UNIV OF TECH
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